3.6.1. Determination of minimal inhibitory concentrations (MICs)

Microorganisms can be tested for their ability to produce visible growth on a series of agar plates (agar dilution), in tubes with broth (broth dilution), or in microplate wells of broth (broth microdilution) containing dilutions of an antimicrobial agent. Additionally, gradient MIC tests are also commercially available. MIC is defined as the lowest antibiotic concentration that prevents visible growth of bacteria. MIC methods are widely used in the comparative testing of new agents, or when a more accurate result is required for clinical management. As there are no CLSI (formerly NCCLS) (www.clsi.org) nor EUCAST (www.eucast.org)  recommendations for the determination of MICs of P. larvae, MIC values of tetracycline and other antibiotics can be determined by the agar dilution method using MYPGP as basal medium (see section 3.1. for recipe) as described as follows:

  1. Obtain antimicrobial powders directly from the manufacturer or from commercial sources.
    The agent must be supplied with a stated potency (mg or International Units per g powder, or as percentage potency).
  2. Store powders in sealed containers in the dark at 4 °C with a desiccant unless otherwise recommended by the manufacturer.
  3. Prepare antibiotic stock solutions by using the following formula:
    Weight of powder (mg) =  Volume of solvent (ml)   X   Concentration (µg/ml)
    Potency of powder (µg /mg)
  4. It is recommended that concentrations of stock solutions should be 1,000 µg/ml or greater.
    In the case of tetracyclines, the tested concentrations can be achieved by using two stock solutions of 5,000 µg/ml and 1,000 µg tetracycline/ml in ethanol, stored at -20°C in darkness until used.
  5. Prepare MYPGP agar flasks and maintain them at 45 °C until the antibiotic solutions are incorporated.
  6. Pour 25 ml of culture medium onto each Petri dish of 90 mm in diameter to give a level depth of 4 mm ± 0.5 mm.
    If using 150 mm diameter Petri dishes, 70 ml of culture medium should be dispensed.
  7. Prepare plates with increasing concentrations of tetracycline i.e.: 0.03, 0.06, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 µg/ml. For the controls, MYPGP agar without antibiotic is used.
  8. Allow the plates to set at RT before moving them.
    Dry the plates in a sterile laminar flow cabinet so that no drops of moisture remain on the surface of the agar; do not over-dry plates.
  9. Incubate each P. larvae strain to be tested on MYPGP agar for 48 h at 36°C to obtain mainly vegetative cells.
  10. Adjust the bacterial suspension until the OD620 (density of a culture determined spectrophotometrically by measuring its optical density at 620 nm)  is about 0.4. 
  11. Each bacterial suspension of each strain must be inoculated onto the surface of the culture medium by adding drops of 5 µl each by means of an automatic micropipette (usually 15-20 drops per plate).
    It is possible to test different strains on the same plate. This procedure must be repeated at least twice for each strain and tetracycline concentration, and control plates without antibiotic must be used. It is strongly recommended to include control strains with known MICs in each batch.
  12. Place the plates open into a sterile laminar flow cabinet until the drops are absorbed.
  13. Incubate the plates in inverted position at 36°C ± 1 for 48 h.
  14. After incubation, ensure that each tested strain has grown on the antibiotic-free plate control.
  15. Read the MIC endpoint for each strain as the lowest concentration of antibiotic at which there is no visible growth.
    The growth of one or two colonies or a fine film of growth should be disregarded.

        Interpretation: for tetracyclines, P. larvae isolates should be considered as ”susceptible” when there MICs are <4 µg/ml, ”intermediate” for MICs  between 4-8 µg/ml and ”resistant”’ for MICs ≥16. Examples of acceptable MIC values  for control strains are: Pseudomonas aeruginosa (ATCC 27853): between 16-32 µg/ml (resistant); Escherichia coli (ATCC 25922): between 0.5-2 µg/ml (susceptible); Staphylococcus aureus (ATCC 29213): between  0.12-1 µg/ml (susceptible) and Enterococcus faecalis (ATCC 29212): between 16-32 µg/ml (resistant).

         When examining a population of bacteria, it is suggested to calculate their values of MIC50 and MIC90 (minimum concentration necessary to inhibit the growth of 50 and 90 % of microorganisms tested respectively).